Shaping the neutrophil: morphological, genomic, and functional maturation

中性粒细胞是数量最多的白色血细胞（每毫升血液中有数百万个），对于保护我们免受病原体的侵害至关重要。同时，中性粒细胞是最短命的白色血细胞（数小时至数天）。因此，骨髓每天需要产生数十亿个中性粒细胞，以保持血液中的数量稳定。当这些数字由于疾病和/或药物治疗而下降时，患者暴露于严重感染的急性威胁。此外，中性粒细胞产生过程中的变化，导致未成熟、功能障碍或过度活跃的中性粒细胞的释放，与许多常见的慢性炎性疾病和癌症有关。.尽管健康的中性粒细胞生成（也称为粒细胞生成）的重要性，但驱动这一过程的分子机制仍知之甚少。在粒细胞生成过程中，祖细胞经历变化和特化，成为成熟的、功能齐全的中性粒细胞。最显著和最广泛的变化之一发生在细胞核上。在多天的过程中，一个简单的圆形核改变形状，变成一个复杂的，由多个裂片组成的分段核。几十年来，细胞核的形状一直被用来指示骨髓中中性粒细胞成熟的程度，但没有人知道为什么或如何形成这种独特的形状。这部分是因为很难将核形状与其他技术提供的中性粒细胞成熟阶段的更详细描述直接联系起来。我们的研究小组已经开发出一种新的方法来更详细地定义细胞核的形状，并在最先进的3D显微镜和人工智能的帮助下在活细胞中进行。在这里，我们建议阐明中性粒细胞核形状的分子编程的第一次。我们怀疑核形状的逐渐变化与粒细胞生成过程中中性粒细胞关键功能的逐渐获得相关，正如我们和其他人之前所建议的那样。因此，对启动和驱动这些壮观变化的分子途径的更好理解将提高我们对粒细胞生成关键过程的理解，以及我们如何操纵或刺激它。细胞核的主要功能是安全而紧密地包装DNA，同时还允许激活DNA中编码的特定基因。一组独特基因的激活决定了细胞的功能和行为。为了更好地了解粒细胞生成过程中观察到的变化背后的分子机制，我们建议在不同程度上研究细胞核。我们将分析DNA区域之间的相互作用，包括相距较远的DNA区域和距离较近的DNA区域。这将标记可能在驱动粒细胞生成中起作用的基因。我们希望首次将核形状，DNA相互作用和中性粒细胞成熟过程中的遗传编程联系起来，以获得粒细胞生成驱动因素的全面了解。我们将使用生物信息学解决方案将这些不同的新数据集联系在一起，以改善解释。接下来，将通过基因编辑技术从中性粒细胞中去除最有希望的粒细胞生成驱动因素。这将使我们能够直接评估编辑细胞的核形状，功能和行为，验证假定驱动程序在粒细胞生成过程中改变核形状和/或中性粒细胞功能的作用。拟议的工作将大大提高我们对中性粒细胞产生的分子编程的理解，这是维持我们健康的关键过程。这将最终弥合现有的零星小鼠和人类数据集，并将开辟更多选择性干扰中性粒细胞功能的可能性。

Neutrophils are the most numerous white blood cell (millions per millilitre of blood) and are crucial for defending us against pathogens. At the same time the neutrophil is the most short-lived white blood cell (hours to days). Hence, billions of neutrophils need to be produced by the bone marrow every day to keep the numbers in the blood stable. When these numbers drop due to disease and/or drug treatments, the patient is exposed to an acute threat of severe infections. Additionally, changes in the neutrophil production process, leading to the release of immature, dysfunctional, or hyperactive neutrophils, have been implicated in many common chronic inflammatory diseases and cancer. . Despite the importance of healthy neutrophil production, also called granulopoiesis, the molecular mechanisms driving this process are poorly understood. During granulopoiesis, progenitor cells go through changes and specializations to become mature, fully functional neutrophils. One of the most notable and extensive changes happens to the cell's nucleus. Over the course of multiple days, a simple, round nucleus changes shape to become a complex, segmented nucleus consisting of multiple lobes. The shape of the nucleus has been used for decades to signal how far the neutrophil maturation in the bone marrow has progressed, yet no-one understands why or how this unique shape is formed. This is partly because it has been difficult to directly link the nuclear shape to the more detailed descriptions of neutrophil maturation stage provided by other technologies. Our research group has developed a novel method to define nuclear shape in much more detail and in live cells, aided by state-of-the-art 3D microscopy and Artificial Intelligence. Here, we propose to elucidate the molecular programming of neutrophil nuclear shape for the first time. We suspect the gradual changes in nuclear shape are correlated to the gradual acquisition of key neutrophil functionalities during granulopoiesis, as has been suggested before by us and others. Hence an improved understanding of the molecular pathways that initiate and drive these spectacular changes will improve our understanding of the crucial process of granulopoiesis and how we might manipulate or stimulate it.The nucleus' main function is to safely and tightly package the DNA, while also allowing the activation of specific genes encoded in the DNA. The activation of a unique set of genes determines cellular functionality and behaviour. To better understand the molecular mechanisms behind the changes seen during granulopoiesis, we propose to study the nucleus at different levels of magnitude. We will analyse interactions between DNA regions both far apart and DNA regions closer together. This will flag the genes that likely have a role in driving granulopoiesis. We expect for the first time to link the nuclear shape, DNA interactions, and genetic programming during neutrophil maturation, to obtain a comprehensive picture of the drivers of granulopoiesis. We will use bioinformatics solutions to tie these different novel datasets together for improved interpretation. Next, the most promising drivers of granulopoiesis will be removed from neutrophils by gene editing techniques. This will allow us to directly assess the nuclear shape, functionality, and behaviour of the edited cells, validating the role of the putative drivers in changing nuclear shape and/or neutrophil function during granulopoiesis.The proposed work will greatly improve our understanding of the molecular programming of neutrophil production, a fundamental process critical to maintaining our health. This will ultimately bridge the existent sporadic mouse and human datasets and will open up possibilities for more selective interference with neutrophil functions.